Comprehensive analysis of NuMA variation in breast cancer

Background A recent genome wide case-control association study identified NuMA region on 11q13 as a candidate locus for breast cancer susceptibility. Specifically, the variant Ala794Gly was suggested to be associated with increased risk of breast cancer. Methods In order to evaluate the NuMa gene for breast cancer susceptibility, we have here screened the entire coding region and exon-intron boundaries of NuMa in 92 familial breast cancer patients and constructed haplotypes of the identified variants. Five missense variants were further screened in 341 breast cancer cases with a positive family history and 368 controls. We examined the frequency of Ala794Gly in an extensive series of familial (n = 910) and unselected (n = 884) breast cancer cases and controls (n = 906), with a high power to detect the suggested breast cancer risk. We also tested if the variant is associated with histopathologic features of breast tumors. Results Screening of NuMA resulted in identification of 11 exonic variants and 12 variants in introns or untranslated regions. Five missense variants that were further screened in breast cancer cases with a positive family history and controls, were each carried on a unique haplotype. None of the variants, or the haplotypes represented by them, was associated with breast cancer risk although due to low power in this analysis, very low risk alleles may go unrecognized. The NuMA Ala794Gly showed no difference in frequency in the unselected breast cancer case series or familial case series compared to control cases. Furthermore, Ala794Gly did not show any significant association with histopathologic characteristics of the tumors, though Ala794Gly was slightly more frequent among unselected cases with lymph node involvement. Conclusion Our results do not support the role of NuMA variants as breast cancer susceptibility alleles.

Fifty-two SNPs were selected for replication genotyping in two independent sample series, one German (188 cases, 150 controls) and one Australian (180 cases, 180 controls) and, among others; a putative breast cancer susceptibility locus was identified at 11q13. The variant with strongest association in the 300 kb region was SNP (rs673478) in an intron of gene LOC220074, p = 0.011 and OR = 1.59 (for combined discovery and replication sets). The high-linkage disequilibrium 300 kb-block was fine-mapped using an additional set of SNPs. The block contains seven annotated genes of which the NuMA gene spanning ~75 kb in the center of the block was chosen as a candidate for breast cancer association. Four SNPs from the fine-mapping SNP set in NuMA were carefully chosen and genotyped in all three sample sets. The strongest association with breast cancer was observed for SNP rs3750913 located in exon 16, which leads to an amino acid substitution Ala794Gly in NuMA protein (p = 0.002, OR = 2.13, for combined sample sets). Furthermore, Kammerer at al. reported an even stronger association for Ala794Gly with familial breast cancer: comparison of frequency in cases with family history of cancer with controls is reported to result in p-value of 0.001 and OR = 4.45 [1].
NuMA (Nuclear Mitotic Apparatus protein) gene is located on chromosome 11q13 and it encodes a 236 kDa nuclear protein essential for normal mitotic spindle organization. NuMA protein consists of globular head and tail domains and a separating long coiled-coil domain [2] which mediates dimerization and oligomerization of NuMA [3]. The tail domain itself is bipartite including a region critical for interaction with the mitotic spindle and another region needed for accurate nuclear reformation including a nuclear localization signal [4][5][6]. Expression of NuMA with deletion in either head or tail domains results in dominant defects in mitosis [7].
NuMA has been associated with acute promyelocytic leukemia (APL). In very rare cases of APL translocation NuMA-RARα (retinoic acid receptor α) t(11;17)(q13;q21) has been observed instead of the common translocation involving a gene fusion of promyelocytic leukemia protein (PML) and RARα [8]. The fusion protein consists of 1883 amino acids of NuMA protein in the aminoterminal end the rest of the total 2285 amino acid protein being derived from RARα -the structure similar to all RARα fusion proteins seen in APL. The essential feature of the fusion partner NuMA is to be capable of establishing protein-protein interactions that may result in formation of defective heterodimers interfering with retinoid signaling [8]. It has been reported that NuMA is an interaction partner of GAS41 (glioma-amplified sequence 41) [9], which is a highly conserved protein and a putative transcription factor amplified in even at early stages of human glioma [10]. This interaction links the structural protein NuMA to the regulation of gene expression. In addition to the previously mentioned studies the role of NuMA has not been under extensive research in association with cancer.
In this study, we screened the NuMA gene for genetic variants in breast cancer cases and studied their relevance for breast cancer risk. In particular, we studied the association of the reported breast cancer susceptibility allele NuMA Ala794Gly in larger series of unselected and familial breast cancer cases and controls, and examined the association of this variant with clinicopathologic characteristics of breast tumors.

Breast cancer patients
The 28 exons and exon-intron boundaries as well as 5' and 3' untranslated regions (ENST00000358965) were screened for sequence variants in a total of 92 familial (non-BRCA1/2) breast cancer cases. Familial breast cancer case was here, in this initial screening, defined as three or more breast cancer cases in first degree family members (including the index case).
A number of identified variants were selected, based on bioinformatic analyses, for further exploratory screening in familial breast cancer patients (n = 341) and healthy population controls (n = 368). The previously identified possible risk variant Ala794Gly was screened in larger series of familial (n = 910) and unselected breast cancer cases (n = 884), and compared in frequency with population controls (n = 906). The unselected breast cancer case series included consecutive newly diagnosed breast cancer patient samples collected at the Department of Oncology, Helsinki University Central Hospital at two separate periods in 1997-1998 and 2000 covering 79% of all newly diagnosed breast cancer cases at respective study periods combined (for more detailed description, see [11,12]. The series of familial breast cancer cases in this study have been collected at the Helsinki University Central Hospital as described in [13]. The successfully genotyped series included 378 patients with strong family history, defined as three or more breast or ovarian cancer cases in the first or second degree family members including the index case. The latter series were screened negative for BRCA1/2 mutations (previously described in detail in [14][15][16]. The remaining 515 successfully genotyped familial cases had a single affected first degree family member; for 312 of these cases, the Finnish BRCA1/2 founder mutations have been excluded as described [14,15]. The cancer diagnoses have been verified through the Finnish Cancer Registry and hospital records. Histopathologic data were collected from pathology reports for all the primary breast tumors available from the unselected case series (889 tumors from 842 successfully genotyped cases). The data set in this study includes information on tumor histology, grade, estrogen and progesterone receptor status, p53 immunohistochemistry, tumor diameter, lymph node status, distant metastases, and the age at the time of diagnosis.
The study was performed with informed consent from the patients and permissions from the Ethics Committee of the Department Obstetrics and Gynecology, as well as from the Ministry of Social Affairs and Health in Finland.

Statistical and Bioinformatic Methods
Standard chi squared or Fisher exact tests were used to assess the differences in genotype frequencies between groups. Per allele odds ratios for each SNP, together with 95% confidence intervals, were estimated using logistic regression. Differences in survival by genotype were assessed using the log-rank test. Analyses were performed using SPSS (SPSS Inc., Chicago, IL). All p-values are twosided. SIFT [17,18] and PolyPhen [19,20] were used for predicting the impact of observed amino acid substitutions on the structure and function of NuMA protein. The most probable haplotypes were reconstructed using the program PHASE [21][22][23].

Results
A total of 11 exonic and 12 intronic or untranslated region (UTR) variants in NuMA were identified (Table 1). We selected 5 of these variants, in addition to Ala794Gly, for further analysis. These five variants had a SIFT score lower than or similar to the score for Ala794Gly (SIFT score 0.29). In particular, two variants (Arg218Trp and Arg1471Trp) had a SIFT score was 0.00, which may indicate that the amino acid substitution is harmful to the protein. We genotyped these variants in 341 familial breast cancer patients and 368 population controls. All were present at a similar frequency in cases and controls.
The Ala794Gly variant was present in 5.8% of population controls (53/906), 5.7% of unselected breast cancer cases (48/842) and 4.8% of familial breast cancer cases (43/ 893) ( Table 3). Thus there was no evidence for an association between Ala794Gly genotype and breast cancer risk.
NuMA Ala794Gly did not show any significant association with histopathologic parameters of the tumors (Table  4). There was some evidence of an association between the Ala794Gly variant and positive lymph node status in unselected breast cancer case series (p = 0.008), but this association was not seen among familial patient series (data not shown). Furthermore, if Bonferroni correction for multiple testing was applied, only p-value of 0.006 or smaller would be considered significant. There was no difference in either overall or disease-free survival by Ala794Gly genotype (data not shown).
Observed variants in NuMA were used for reconstructing haplotypes using PHASE program (Table 5). Altogether 15 different haplotypes defined by 23 sequence variants in NuMA were observed in the screening of 92 familial breast cancer patients ( Table 5). The most common haplotype (without any observed DNA sequence variants) accounts for 80% of observed haplotypes, and together the three most common haplotypes account for 90% of haplotypes. Each of the missense variants that were further studied represent a unique haplotype except the Ala794Gly which is present in two haplotypes (numbers 12 and 14 in Table 5) defined by an intronic variant IVS2+34 G > C. In order to study the possibility that either of haplotypes 12 or 14 would associate with breast cancer risk we screened the haplotype-defining intronic variant IVS2+34G > C in 337 familial breast cancer cases and 359 controls. Variant IVS2+34G > C was present in cases and controls in similar proportions (cases 49/337, 14.5% and controls 57/359, 16.0%; p = 0.6). Furthermore, we did not observe any haplotype 12 carriers in this further screening (variant IVS2+34G > C was always present in Ala794Gly carriers).

Discussion
Kammerer et al. utilized a genome-wide association analysis to identify breast cancer susceptibility regions and identified, among other regions, a high-linkage disequilibrium region on chromosome 11q13 [1]. This region contains several genes and NuMA was chosen as a most  likely candidate for breast cancer susceptibility gene, and variant Ala794Gly was hypothesized to be functionally impaired and suggested to be associated with breast cancer risk [1].
Our thorough screening of NuMA gene in breast cancer cases resulted in identification of several variants of which eight were missense changes and the rest were synonymous variants or not located in coding regions. Missense variants that warranted further screening after bioinformatic analyses were present in the breast cancer cases in similar frequencies as in controls. Variant Arg1471Trp was not detected in controls, however, but being present in only 0.6% of the cases would have only marginal effect on breast cancer even if having any effect on breast cancer risk for the carriers. These variants represented also unique haplotypes, not suggesting presence of other risk alleles in linkage disequilibrium with these variants either, although due to low power, very low risk alleles may go unrecognized.
We also specifically evaluated the variant NuMA Ala794Gly for breast cancer risk in an extensive patient series (884 unselected cases, 910 familial cases and 906 population controls) as compared to those used by Kammerer et al, (2005) [1]. For this analysis, our material has 98% power to detect a difference in frequency of that magnitude.
Variant Ala794Gly was detected in almost equal frequencies in all our study series as well as in population controls, which is consistent with the results from a large The mean age of diagnosis was 57 years for C:C, and 56 for C:G genotypes.
breast cancer patient and control series recently studied by the Breast Cancer Association Consortium [24] and does not support the previously proposed association with breast cancer risk. However, as the variant Ala794Gly was initially found here to be present in two distinct haplotypes defined by an intronic variant it is possible that differences in the relative haplotype frequencies could have masked any associated risk. Further evaluation of these two haplotypes, however, did not support this possibility. None of the identified NuMA variants was associated with breast cancer risk in our study. Furthermore, Ala794Gly variant was not significantly associated with any of the tumor characteristics.

Conclusion
In conclusion, our results do not support the role of NuMA variants as breast cancer risk alleles.